This application contains subject matter which is related to that in patent application Ser. No. 266,278 entitled "Method and Apparatus for Detection and Purification of Proteins and Antibodies" filed June 26, 1972, now abandoned; Ser. No. 392,950 entitled "Method For Binding Antibodies To a Surface Such That They Remain Active" filed Aug. 30, 1973 now abandoned; and Ser. No. 392,951 entitled "Method for Improving Contrast in Surface Immunological Tests with Large Size Proteins" filed Aug. 30, 1973, now abandoned all having a common applicant and assignee.
Other publicantions related to the present invention primarily as background are "Optical Measurement of the Thickness of a Film Adsorbed from a Solution", authors Irving Langmuir et al. Journal of the American Chemical Society, Vol. 59 (July-December 1937) page 1406; "Immunological and Enzymatic Reactions Carried Out at a Solid-Liquid Interface" by Alexandre Rothen, Physiological Chemistry and Physics, 5, (1973), pages 243-258; "Interactions Among Human Blood Proteins at Interfaces", Leo Vroman et al, Federation Proceedings, Vol. 30, No. 5 (September-October 1971) pages 1494-1502; and "The Antibody-Antigen Reaction: A Visual Observation", Ivar Giaever, The Journal of Immunology, Vol. 110, No. 4 (May 1973) pages 1424-1425.
Immunological reactions are highly specific interactions in which an antigen interacts with a corresponding second biological constituent specific to the antigen and generally known as the antibody to form an immunological complex. Immunological reactions taking place within a biological system such as an animal or a human being are vital in combating disease. In a biological system, the entry of a foreign biological constituent, e.g., the antigen, causes the biological system to produce the specific antibody to the antigen in a process not fully understood at this time. The antibody molecules have available chemical combining or binding sites which complement those on the antigen molecule so that the antigen and antibody combine or bond to form the immunological complex.
Antibodies are produced by biological systems in response to invasion thereof by foreign bodies. Consequently, the detection of antibodies present in a biological system is of medical diagnostic value in determining the antigens to which the system has been exposed. It would be useful, for example, to test for the presence of the antibody to syphilis or gonorrhea in human blood, plasma or tissue. Conversely, the detection of certain antigens in a biological system also has medical diagnostic values; examples of diagnostic detection of antigens include detection of human chorionic gonadotrophin (HCG) protein molecules in urine as a test for pregnancy, and detection of hepatitis-associated antigen (HAA) molecules in blood of prospective blood donors.
Most antigens are proteins or contain proteins as an essential part, whereas all antibodies are proteins. Proteins are large molecules of high molecular weight, i.e., are polymers consisting of chains of amino acids. The antigen and antibody protein may each have several combining sites. The five major classes of antibodies (immunoglobulins IgG, IgM, IgA, IgE and IgD are each apparently characterized by at least two heavy (long) peptide chains of amino acids and at least two light (short) peptide chains of the acids wherein the bond between the amino acids units is known as a peptide bond. These heavy and light peptide chains are oriented in the general shape of the letter "Y" and the active or combining sites are the extreme ends of the two arms of the Y-shaped antibody for the IgG antibody.
In addition to the immunological reaction which occurs between specific protein antigens and specific protein antibodies resulting in the formation of a protein antigen-protein antibody complex, other immunological complexing reactions between immunologically reactive antigens and antibodies are also contemplated by this invention. In addition, specific reactions between other biological particles, such as enzymes and their substrates, are also among the methods contemplated and are embraced by the term "immunological reaction" as used herein. Furthermore, as used herein, the terms "antigen" and "antibody" are meant to encompass such terms as enzymes, substrates, and similar biological particles. As will also be seen, the method is versatile enough to permit substitution of a specific antibody for the corresponding antigen and the antigen for the corresponding specific antibody.
For instance, the following systems include biological particles which are capable of undergoing the immunological reactions described herein:
Viruses PA1 Bacteria and Bacterial toxins PA1 Fungi PA1 Parasites PA1 Animal tissue PA1 Animal body fluids, and the like. PA1 trypsin extract -- trypsin antibody PA1 chymotrypsin extract -- chymotrypsin antibody PA1 pepsin extract -- pepsin antibody PA1 ribonuclease extract -- ribonuclease antibody PA1 thrombin extract -- thrombin antibody PA1 amylase extract -- amylase antibody PA1 penicillinase extract -- penicillinase antibody PA1 insulin -- insulin antibody.
With respect to viruses, the antigens are viral cultures, or parts thereof, and the antibody specific thereto can be produced by administration to a living host. Illustratively, antigen-antibody complexes in the following virus systems are useful in the hereindisclosed procedure: Rubella virus culture (antigen) -- Rubella virus antibody; polio virus culture (antigen) -- polio virus antibody; vesicular stomatitis virus (VSV) culture (antigen) -- VSV antibody.
Regarding bacteria and bacterial toxins, the antigens are the particular bacteria or bacterial toxin, or parts thereof, and the antibody is produced by injection into a living host. The following are illustrative examples of antigen-antibody pairs which can be used in the present method: tetanus toxoid suspension (antigen) -- tetanus antibody; diptheria toxin suspension (antigen) -- diptheria antibody; Neisseria gonorrhoea suspension (antigen) -- gonorrhea antibody; Treponema palladium suspension (antigen) -- syphilis antibody.
As for fungi, the antigens are antigenic extracts of fungal suspensions and the antibody is the fungal antibody produced by injection into a living host. Antigen-antibody complexes of fungi systems are illustrated by the following: Aspergillus extract suspension (antigen) -- aspergillus fungus antibody; Candida extract suspension (antigen) -- candida fungus antibody.
Antigens and antibodies in parasite systems are tested in a similar fashion to those of fungi. The system Toxoplasma gondii extract (antigen) -- Toxoplasma gondii antibody is a typical example.
By the term polysaccharides is meant a system wherein the antigen is a carbohydrate antigen. An example of such an antigen-antibody containing system is pneumococcus polysaccharides (antigen) -- pneumococcus antibody.
In addition to the typical enzyme-enzyme substrate reaction which is intended to be covered herein, enzymes themselves, or parts thereof, may be utilized as antigens, and the antibody is the particular enzyme antibody elaborated by a living host after injection. Illustrative antigen-antibody complexes of enzyme systems are:
With respect to hormones, the antigenic constituent is usually found in a hormone extract and the antibody is the particular hormone antibody elaborated by the living organism after injection. An exemplary antigen-antibody complex is:
Although the ensuing discussion is directed for the most part to immunological interactions between specific protein antigens and specific protein antibodies, it is understood that it also applied to the systems and the immunologically reactive antigens and antibodies hereinabove described.
As presently practiced, the diagnostic utilization of immunologically active antibodies and antigens relay upon the precipitating or agglutinating characteristic thereof resulting from the immunological complexing reaction. The classic example of these diagnostic uses is the blood typing procedure in which blood samples are mixed with alpha and beta type serum antibodies and blood type is determined by observing any agglutination occurring in the blood samples.
The HCG pregnancy test as currently practiced is an inhibition test. The test is performed by mixing a quantity of HCG anti-serum into a urine specimen. A plurality of polystyrene spheres which have been coated with HCG protein are then introduced into the previously prepared urine specimen. The polystyrene spheres will agglutinate if, but only if, HCG protein is absent from the urine specimen. If HCG protein is absent from a urine specimen, the HCG protein on the polystyrene spheres complexes with the HCG anti-serum previously introduced in the urine specimen and the spheres agglutinate. If, on the other hand, HCG protein is present in sufficient quantities in the urine specimen, it complexes with the previously introduced HCG anti-serum forming a complex which precipitates out of the specimen so that the previously introduced anti-serum is no longer available to complex with the HCG protein on the spheres to cause agglutination thereof. In accordance with the teachings of this disclosure, the present HCG protein pregnancy test could be simplified by adhering HCG anti-serum onto the polystyrene spheres and directly testing a urine specimen. In this case, the polystyrene's spheres would agglutinate if, but only if, HCG protein is present in the specimen.
It appears that the reason this simpler procedure has not been employed is that the available HCG anti-sera are complex mixtures containing a large proportion of constituents other than HCG antibodies. The additional effort required in the prior art to extract the antibodies from the HCG anti-sera make the inhibition test, utilizing sera directly, preferable in the prior art. However, in accordance with one embodiment of this invention, a procedure is provided whereby the simpler, direct test is performable. A serious shortcoming of agglutination tests overcome by the present direct method is that the particles involved may tend to agglomerate for any of a variety of reasons having nothing to do with immunological agglutination thereby decreasing the reliability of the test. Typically, even though agglutination tests are performed with great care by skilled technicians, nevertheless occasional diagnostic errors occur.
Even though it is known that the antibody-antigen complexing reaction will take place when an antigen (or antibody) is adsorbed at a surface, the technique has not been widely applied to routine medical diagnoses on slide like substrates. The complexing reaction at a surface has been observed by means of an ellipsometer. An ellipsometer is a complex optical instrument by means of which it is possible to measure the thicknesses of films on the order of 0.1 A. Ellipsometers are expensive and require skilled operators. In studies of immunological reactions using ellipsometers performed to date, two methods have been used. In one method, the reaction to be studied is allowed to take place and then the slide on which the reaction has taken place is mounted in an ellipsometer and an actual measurement of film thickness is made. In the other method, the slide is mounted in the ellipsometer while the immunological reaction is taking place and the change of film thickness is observed with the ellipsometer. The measurement of absolute thickness requires extreme care. On the other hand, when the concentration of antibodies in the solution is low, the measurement of absolute thickness will take a long time. Accordingly, for practical reasons, the detection of immunological reactions at a surface using an ellipsometer has not been adopted for diagnostic purposes.
The present invention is based on the discovery that any arbitrary immunologically reactive antigen will adsorb onto a substrate in a monomolecular layer only, and that a specific antibody for such arbitrary antigen will bond thereto to form a bimolecular layer on the substrate. In practicing this invention, such layers are easily and quickly detectable. This discovery then provides methods and apparatus of great utility in medical diagnostic and pharmacological applications.
Accordingly, it is a first principal object of this invention to provide method and apparatus for economically detecting immunological reactions occurring at a surface.
It is an object of this invention to provide such method and apparatus wherein such immunological reactions are detectable by electrical means.
It is another object of this invention to provide such method and apparatus for detecting such immunological reactions by direct visual observation.
A second principal object of this invention is to provide method and apparatus for detecting the presence or absence of antigens and antibodies by means of controlled immunological reactions occurring at a surface.